Over the past 26 months our laboratory has investigated G-protein expression and AC activity in alcoholics, nonalcoholics and the nonalcoholic family members of both groups. Our 'work in progress' demonstrates differential expression of Gsalpha levels in nonalcoholic individuals with a family history of alcoholism [family history positive (FHP) compared to nonalcoholic individuals without a family history of alcoholism (family history negative (FHN)]. These data suggest an intrinsic abnormality in the AC system of alcoholics and those biologically vulnerable to this disorder. In our renewal, we shall confirm our important observation that FHP nonalcoholic subjects have enhanced expression of Gsalpha in erythrocyte and lymphocyte membranes by increasing the sample size and including female subjects. We will examine cell lines from control, alcoholic, and unaffected children of alcoholic subjects. From the promising elements, we shall examine the particularly informative stage IV families for appropriate linkage analysis. We shall investigate the potential significance of differential expression of Gsalpha in FHP and FHN nonalcoholic subjects by correlating membrane G-protein expression/function (conducted at JHU) with sophisticated studies of tolerance/activation. The significance of membrane Gsalpha levels in the development of tolerance will be pursued utilizing two manipulatable models of G-protein expression. In the first model, we will employ several stable cell lines created from S49 cells which were designed to have varying degrees of Gsalpha expression. These cell lines recapitulate the spectrum of G-protein expression in humans. We shall test the hypothesis that the degree of ethanol-induced activation/tolerance in the AC system is proportional to amount of Gsalpha expression. We will also utilize a transgenic mouse model of enhanced Gsalpha expression to determine the effects of varying amounts of cellular Gsalpha on the acquisition of tolerance on the whole animal level. Lastly, we shall continue our studies determining if genetic linkage exists between the inheritance of the alcoholism phenotype and specific Gsalpha gene alleles. To date, our analysis has allowed us to detect several polymorphisms in the Gsalpha. We shall obtain DNA from cell lines of control, alcoholic, and unaffected children of alcoholic subjects. From the promising elements, we shall examine the particularly informative stage IV families for appropriate linkage analysis.